Principle: Proteins are captured on the bottom of a micro well plate, either by direct binding or by a conjugated antibody "trap". A second antibody is added to detect one specific type of protein. A counter-stain antibody (usually HRP-conjugated) is used to generate visible signal, which is proportional to the number of proteins. Normalization (e.g., using the number of cells per lysate sample, and a purified protein with known concentration if you're fortunate to have one available) can be used to calculate proteins per cell.

DIRECT ELISA, HRP DETECTION

This key feature of this approach is the attachment of proteins directly to the bottom surfaces of the micro wells. A specific protein is detected with a primary and secondary-HRP. The advantage over "sandwich" ELISA is that fewer antibodies are needed, but this method is known to be less sensitive/ accurate. Because of this I recommend this method for artificially over-expressed or abundant naturally expressed proteins.

Washing: Fill each well with '1X femto‐TBST (~350μl) and wait for 30 seconds then invert the wells to empty and gently tap out the residual liquid from each well. Repeat the washing procedure 4‐5 times.

Secondary antibody-HRP

Add 100μl HRP‐labeled secondary antibody solution (diluted in 1x NAP‐Blocker) to each well and incubate for 1 hour at room temperature. After incubation, empty the plate and gently tap out the residual liquid.

Washing: Fill each well with 1x femto‐TBST (~350μl) and wait for 30 seconds then invert the plate to empty and tap out the residual liquid from each well. Repeat the above washing steps 4‐5 times.

Finally add 350μl of 1x femto‐TBST into each well and wait for 5 minutes. Tap out the residual wash from each well and plate is ready to develop with femtoELISATM HRP Substrate.

Signal detection

Add 100μl of femtoELISA HRP Substrate into each well. A soluble blue color develops, which can be read at 370nm or at the 620nm to 650nm range, using femtoELISATM HRP Substrate as a blank.

What to do with your data: calculate unknown protein concentration(s) per cell